U.S. patent number 4,916,224 [Application Number 07/146,252] was granted by the patent office on 1990-04-10 for dideoxycarbocyclic nucleosides.
This patent grant is currently assigned to Regents of the University of Minnesota. Invention is credited to Mei Hua, Robert Vince.
United States Patent |
4,916,224 |
Vince , et al. |
April 10, 1990 |
**Please see images for:
( Certificate of Correction ) ** |
Dideoxycarbocyclic nucleosides
Abstract
Antiviral and antitumor compounds are disclosed of general
formula: ##STR1## wherein Z is H, OH or NH.sub.2, Y is CH or N, the
bond indicated by C.sub.1 '--C.sub.2 ' is absent or, in combination
with the C.sub.1 '--C.sub.2 ' bond is the unit CH.dbd.CH, and X is
selected from the group consisting of H, N(R.sub.2), SR, OR or
halogen, wherein R is H, lower (C.sub.1 -C.sub.4)alkyl, aryl or
mixtures thereof, and the pharmaceutically acceptable salts
thereof.
Inventors: |
Vince; Robert (St. Paul,
MN), Hua; Mei (Minneapolis, MN) |
Assignee: |
Regents of the University of
Minnesota (Minneapolis, MN)
|
Family
ID: |
22516514 |
Appl.
No.: |
07/146,252 |
Filed: |
January 20, 1988 |
Current U.S.
Class: |
544/254; 544/264;
544/267; 544/277; 544/265; 544/276 |
Current CPC
Class: |
C07D
239/50 (20130101); C07D 487/04 (20130101); C07D
239/48 (20130101); C07D 473/00 (20130101) |
Current International
Class: |
C07D
487/04 (20060101); C07D 487/00 (20060101); C07D
473/02 (); C07D 473/26 (); C07D 473/40 (); A61K
031/52 () |
Field of
Search: |
;544/254,276,277,265,267,264 ;514/261,262,258 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
236935 |
|
Sep 1987 |
|
EP |
|
0219284 |
|
Dec 1984 |
|
JP |
|
Other References
Santi et al., Chemical Abstracts, vol. 76:22492h (1972). .
Thomas et al., Chemical Abstracts, vol. 68:49934a (1968). .
Santi et al., Chemical Abstracts, vol. 78:132248g (1973). .
Coward et al., Chemical Abstracts, vol. 79:87815x (1973). .
S. Broder, ed., AIDS-Modern Concepts and Therapeutic Challenges,
Marcel Dekker, Inc., NY (1987), p. 320. .
J. P. Horwitz et al., J. Org. Chem., 32, 817 (1967). .
J. Balzarini et al., Biochem. Biophys. Res. Comm., 140, 735 (1986).
.
T.-S. Lin et al., Biochem. Pharmacol., 36, 311 (1987). .
T.-S. Lin et al., J. Med. Chem., 30, 440 (1987). .
P. Herdewijn et al., J. Med. Chem., 30, 1270 (1987). .
J. Balzarini et al., Molec. Pharmacol., 32, 162 (1987). .
M. Baba et al., Biochem. Biophys. Res. Commun., 142, 128 (1987).
.
H. Lee and R. Vince, J. Pharm. Sci., 69, 1019 (1980). .
R. Vince et al., J. Med. Chem., 27, 1358 (1984). .
R. Vince et al., J. Med. Chem., 20, 612 (1977). .
L. B. Townsend, in Nucleoside Analogues-Chemistry, Biology and
Medical Applications, R. T. Walker, E. DeClerq and F. Eckstein,
eds., Plenum Press, NY (1979), pp. 193-223. .
R. Vince et al., Science, 221, 1405 (1983). .
Shealy et al., J. Pharm. Sci., 62, 1432 (1973). .
H. J. Schaeffer et al., J. Pharm. Sci., 53, 1510 (1964). .
Y. F. Shealy et al., J. Heterocycl. Chem., 10, 601 (1973)..
|
Primary Examiner: Shah; Mukund J.
Assistant Examiner: Rivers; Diana G.
Attorney, Agent or Firm: Merchant, Gould, Smith, Edell,
Welter & Schmidt
Government Interests
GRANT INFORMATION
This invention was made with Government support under Grant No. 5 R
01 CA23263, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
What is claimed is:
1. A compound of the formula: ##STR7## wherein X is OH NH.sub.2 or
Cl, Z is NH.sub.2 and Y is CH or N, and the pharmaceutically
acceptable salts thereof.
2. The compound of claim 1 wherein X is Cl.
3. The compound of claim 1 wherein X is OH.
4. The compound of claim 1 wherein X is NH.sub.2.
5. A compound of the formula: ##STR8## wherein Z is H, OH or
NH.sub.2, Y is CH or N, and X is selected from the group consisting
of H, N(R).sub.2, SR, OR and halogen, wherein R is H, lower(C.sub.1
-C.sub.4)alkyl, aryl or mixtures thereof, and the
pharmaceutically-acceptable salts thereof.
6. The compound of claim 5 wherein Y is CH.
7. The compound of claim 6 wherein Z is H.
8. The compound of claim 7 wherein X is Cl.
9. The compound of claim 7 wherein X is NH.sub.2.
10. The compound of claim 7 wherein X is SH.
11. The compound of claim 6 wherein Z is NH.sub.2.
12. The compound of claim 11 wherein X is Cl.
13. The compound of claim 11 wherein X is OH.
14. The compound of claim 11 wherein X is NH.sub.2.
Description
FIELD OF THE INVENTION
The present invention relates to dideoxycarbocyclic nucleosides
which exhibit antiviral and cytotoxic activity.
BACKGROUND OF THE INVENTION
Despite intensive effort to discover drugs that may be of value in
the systemic treatment of human immunodeficiency virus (HIV)
infections, such infections have been singularly resistant to
chemotherapy. The intracellular and intimate relation to nuclear
metabolism of virus reproduction makes it difficult to destroy a
virus without irreparable damage to the host cell.
The discovery of the antiviral activity of vidarabine
(9-.beta.-D-arabinofuranosyladenine monohydrate) has led to the
preparation of a large number of synthetic nucleosides. To date,
only one synthetic nucleoside, 3'-azido-3'-deoxythymidine has been
approved for treating certain AIDS patients, but it is a pallative,
not a cure. ##STR2##
Although AZT is specifically active against retroviruses, its use
has lead to side effects, including anemia, headache, confusion,
anxiety, nausea and insomnia. The nucleoside analog,
2'-3'-dideoxycyfide (DDC), exhibits an in vitro TI.sub.50 of ca.
300 against HIV and may exhibit fewer side effects than AZT, but
may also be eliminated more rapidly from the body. ##STR3## The
synthesis of adenine ("6-amino-purine") nucleoside analogs in which
the pentose sugar has been replaced with tris(hydroxy)-substituted
cyclopentyl residues has yielded compounds with substantial
cytotoxic and antiviral activity. For example, the carbocyclic
analog of vidarabine, cyclaridine, is highly active against HSV-2,
but exhibits a low therapeutic index (TI.sub.50 =10) against HIV in
vitro. Likewise, the carbocyclic analog of AZT is inactive against
HIV. Therefore, it is clear that the structure-activity
relationships between the variously substituted carbocyclic
nucleosides which have been prepared and tested remain
ill-defined.
Thus, a substantial need exists for chemotherapeutic agents
effective to protect mammalian cells against infection by viruses
such as HSV-2, HIV, varicella-zoster, vaccinia, human
cytomegalovirus (HCMV) and the like.
SUMMARY OF THE INVENTION
The present invention is directed to hydroxymethyl-cyclopentyl or
-cyclopentenyl-substituted purines and 8-aza-purines of the formula
(I): ##STR4## wherein Z is H, OH or NH.sub.2, Y is CH or N, the
bond indicated by--is either absent or, in combination with the
C.sub.1 '-C.sub.2 ' bond, is the unit CH.dbd.CH and X is selected
from the group consisting of H, N(R).sub.2, SR, OR and halogen,
wherein R is H, lower(C.sub.1 -C.sub.4)alkyl, aryl or mixtures
thereof, and the pharmaceutically acceptable salts thereof.
Preferably, X is Cl, OH or NH.sub.2, Y is CH, R is phenyl or H, and
the bond indicated by--is present. Certain of these compounds are
effective antiviral and/or cytotoxic agents or are intermediates
useful for the preparation thereof.
Although generally, compounds of formula I are not active against
HSV-1, it is expected that some of them will exhibit specific
antiviral activity against other viruses such as HSV-2, HCMV and/or
HIV. Specifically, the compound of formula I, wherein X is OH, Z is
NH.sub.2, Y is CH and the bond--is present, (14a) strongly inhibits
HIV infectivity in vitro. The TI.sub.50 of this compound varied
with the infected cell line which was used to assay for anti-HIV
activity, but generally fell between 200-400, and was determined to
be as high as 667 in one assay. Compound 14a is also active against
HSV-1. Compounds of formula I wherein X is Cl or NH.sub.2, Y is CH,
Z is NH.sub.2 and the bond--is present (13a and 15a, respectively)
are also active against HIV. Compounds of the formula I wherein
Y.dbd.N, Z.dbd.NH.sub.2, X.dbd.Cl or OH and the bond indicated
by--is either absent or present, are cytotoxic to cultured P-388
leukemia cells.
It is believed that the antiviral activity is due to an inhibitory
effect on the ability of viruses to infect normal mammalian cells.
The present invention is also directed to the intermediate compound
of the formula (II): ##STR5## wherein Z is H or NH.sub.2, Z' is H
or NH.sub.2, X is halogen, preferably Cl, and the bond indicated
by--is either absent or, in combination, with C.sub.1 '-C.sub.2 '
bond, is the unit CH.dbd.CH, which is useful for the preparation of
the purines of the invention. Preferably, Z is NH.sub.2, and Z' is
H or both Z and Z' are NH.sub.2. However, the compounds where
X.dbd.Cl, Z.dbd.NH.sub.2 and Z'.dbd.H or NH.sub.2 are not active
against HIV.
The (3-hydroxymethylcyclopentenyl)pyrimidine analog, 20a, is also
within the scope of the present invention. Its synthesis from
cyclopentene 2a is outlined in Scheme I, below. ##STR6##
Thus, it is expected that certain of the compounds of the present
invention will be useful against viral infections or
virus-associated tumors, and the method of their use to inhibit
viral infectivity or tumor growth in vitro or in vivo is also
within the scope of the present invention. More specifically, the
2',3'-dideoxyadenosine compounds of formula I, wherein Z is H, X is
Cl, OH, NH.sub.2 or SH, Y is CH and the C.sub.1 '-C.sub.2 ' bond is
absent have been previously reported by H. J. Schaeffer et al., J.
Pharm. Sci., 53, 1510 (1964), and been disclosed to be not useful
against HIV. See AIDS-Modern Concepts and Therapeutic Challenges,
S. Broder, ed., M. Dekker, Inc., New York, N.Y., (1987) at pages
316-317. Likewise, compound I, wherein Z is H, X is NH.sub.2, Y is
N and the C.sub.1 '-C.sub.2 ' bond is absent has been reported by
Y. F. Shealy et al., J. Heterocycl. Chem., 10, 601 (1973), but has
not been disclosed to be active against HIV.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a flow diagram summarizing the synthesis of the purines
of the present invention.
FIG. 2 is a graphic depiction of cells exposed to 14a/control cells
(%) plotted vs. concentration of 14a for both uninfected cells and
cells infected with HIV.
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 outlines the synthesis of preferred compounds of formulas I
and II from starting materials 1a and 1b. The structural formulas
and some of the properties of compounds 7a-18a and 7b-18b are
summarized on Table I, below. T1 TABLE I-A.
2',3'-Dideoxy-6-Substituted-Purines of Formula I, - Z = H. -Com-
-pound Yield -No. X Y (C.sub.1 '-C.sub.2 ')* M.P. (.degree. C.) Rf
(%) -7a Cl CH + 108-110 0.35.sup.a 82 -7b Cl CH - 120-122
0.33.sup.a 86 -8a OH CH + 248-250(dec) 0.24.sup.b 45 -8b OH CH -
276-278(dec) 0.23.sup.b 60 -9a NH.sub.2 CH + 198-200 0.33.sup.b 81
-9b NH.sub.2 CH - 146-148 0.30.sup.b 77 -10a SH CH + 263-265(dec)
0.44.sup.b 73 -10b SH CH - 297-300(dec) 0.42.sup.b 69 -11a OH N +
180-182 0.38.sup.b 49 -11b OH N - 179-182 0.37.sup.b 69 -12a
NH.sub.2 N + 220-222(dec) 0.45.sup.b 69 -12b NH.sub.2 N - 195-196
0.44.sup.b 79 -B. 2'-3'-Dideoxy-2,6-Disubstituted-Purines of -
Formula I, Z = NH.sub.2. -Com- -pound Yield -No. X Y (C.sub.1
'-C.sub.2 ')* M.P. (.degree. C.) Rf.sup.b (%) -13a Cl CH + 145-147
0.64 80 -13b Cl CH - 151-153 0.62 73 -14a OH CH + 254-256(dec) 0.27
61 -14b OH CH - 274-276(dec) 0.25 77 -15a NH.sub.2 CH + 152-155
0.41 80 -15b NH.sub.2 CH - 208-211 0.39 77 -16a Cl N + 153-155(dec)
0.69 81 -16b Cl N - 170-172(dec) 0.67 82 -17a OH N + 223-225(dec)
0.40 89 -17b OH N - 228-231 0.38 81 -18a NH.sub.2 N + 240-242(dec)
0.52 83 -18b NH.sub.2 N - 223-225 0.50 88 -
Compounds 7a, 7b, 8b, 9a, 9b, 10a, 10b, 13a, 14a, 14b, 15a, and
15b, are effective in the anti-HIV assay described in Example 36.
Likewise, compounds 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 19a and 20a,
shown on FIG. 1, are effective to inhibit the infection and killing
of human T lymphocytes (T.sub.h cells) by HIV. Therefore, these
compounds are candidates for clinical trials in human patients
infected with HIV and/or afflicted with AIDS or AIDS-related
complex (ARC).
DETAILED DESCRIPTION OF THE INVENTION
The synthesis of the hydroxymethylcyclopentenyl compounds of
formula 7a-18a and the hydroxymethylcyclopentyl compounds of
formulas 7b-18b, from the versatile precursor,
1.alpha.-acetylamino-3.alpha.-acetoxymethylcyclopent-2-ene (1a) was
accomplished as outlined in FIG. 1. Compound 1a was prepared as
described in U.S. Pat. No. 4,138,562, the disclosure of which is
incorporated by reference herein. Compound 2a was prepared from
compound 1a by hydrolysis in the presence of a mild base, such as
an alkaline earth metal hydroxide. To afford the pyrimidine
compound 3a, compound 2a was reacted with an excess of
5-amino-4,6-dichloropyrimidine in the presence of an amine base,
such as a trialkylamine, in an alcoholic solvent. Likewise, the
cyclopentanyl compound 1b, which is obtained from compound 1a by
hydrogenation, is hydrolyzed and reacted with
5-amino-4,6-dichloropyrimidine to yield the pyrimidinylcyclopentyl
carbinol, 3b. Also, 2-amino-4,6-dichloropyrimidine was reacted with
compound 2a to yield compound 4a, and with compound 2b to yield
compound 4b.
Para-chloroaniline was diazotized with acidic sodium nitrite and
reacted with compound 4a and 4b to yield the chlorophenylazo
intermediates 5a and 5b, respectively. Reduction of the azo
intermediates 5a and 5b to yield 6a and 6b, respectively, was
accomplished with zinc and acetic acid. See Shealy and Clayton, J.
Pharm. Sci., 62, 1433 (1973).
The 5-amino-6-chloro-4-pyrimidinyl intermediates 3a and 3b were
converted to the 9-substituted-6-chloropurines 7a and 7b,
respectively, by ring closure with triethylorthoformate and
subsequent mild acid hydrolysis to remove ethoxymethylidenes and
formates formed during the reaction. In like manner, the
2,5-diamino-6-chloro-4-pyrimidinyl intermediates 6a and 6b were
ring-closed to the corresponding 2-amino-6-chloro-9H-purin-9-yl
compounds 13a and 13b.
The 6-chloropurines 7a, 7b, 13a and 13b were converted to the
corresponding 6-hydroxy purines 8a, 8b, 14a and 14b, respectively,
with aqueous base, i.e., by refluxing them with an alkali metal
hydroxide such as NaOH. Chloro compounds 7a, 7b, 13a, 13b, 16a and
16b were converted to the corresponding amino compounds 9a, 9b,
15a, 15b, 18a and 18b, by reaction with liquid ammonia under
pressure.
Mono- or disubstituted 6-amino compounds of formula I, wherein X is
NR.sub.2 and R=R=(lower)alkyl, phenyl or mixtures thereof with H
can be prepared by conventional methods for the conversion of
halides to secondary or tertiary amines. For example, see I. T.
Harrison et al., Compendium of Organic Synthetic Methods,
Wiley-Interscience, N.Y. (1971) at pages 250-252. The 6-chloro
substitutent in compounds 7a, 7b, 13a, 13b, 16a and 16b can be
replaced with other halogen atoms by the use of various
p-(halo)benzene diazonium chlorides in the conversion of 4a to 5a
or of 4b to 5b, or by conventional methods of halide-halide
exchange.
These conversions are extensively described in the context of
purine nucleoside synthesis in Nucleoside Analogs-Chemistry,
Biology and Medical Applications, R. T. Walker et al., eds., Plenum
Press, N.Y. (1979) at pages 193-223, the disclosure of which is
incorporated by reference herein.
Treatment of 7a and 7b with thiourea in refluxing alcohol, followed
by alkaline hydrolysis afforded thiols 10a and 10b, respectively.
See L. F. Fieser et al., Reagents for Organic Synthesis, John Wiley
and Sons, Inc., N.Y. (1967) at pages 1165-1167 and U.S. Pat. No.
4,383,114, the disclosures of which are incorporated by reference
herein. Phenyl or alkylthioderivates can be prepared from the
corresponding thiols by the procedure of U.S. Pat. No. 4,383,114
(Example 6).
Ring closure of 3a and 3b with acidic aqueous sodium nitrate
followed by neutralization with aqueous base directly afforded the
corresponding 7-hydroxy-3H-1,2,3-triazolo[4,5d]pyrimidin-3-yl
compounds 11a and 11b, respectively. Ring closure of 6a and 6b
afforded the corresponding
5-amino-7-chloro-3H-1,2,3-triazo[4,5-d]pyrimidin-3-yl compounds 16a
and 16b, respectively, which were hydrolyzed to the corresponding
7-hydroxy compounds 17a and 17b with aqueous NaOH. Compound 3a was
converted to the corresponding 7-amino compounds 12a by reaction
with acidic sodium nitrite, followed by reaction of the crude
product with liquid ammonia. The 7-aminocyclopentyl carbinol 12b
was prepared by hydrogenating 12a (Pd-C). Compounds of formula I,
wherein Z is OH, X is NH.sub.2 or OH, and Y is CH can be prepared
from compounds 14a, 14b, 15a or 15b by deamination of the 2-amino
group with nitrous acid, employing the procedure used by Davoll to
convert 2-aminoadenosine to isoguanosine. See J. Davoll, J. Amer.
Chem. Soc., 73, 3174 (1951), the disclosure of which is
incorporated by reference herein.
Compounds of formula I, wherein X is H, Z is NH.sub.2 and Y is CH
can be prepared from compounds 7a, 7b, 13a or 13b by dehalogenation
with zinc/water [J. R. Marshall et al., J. Chem. Soc., 1004 (1951)]
or by photolysis in dry nitrogen-purged tetrahydrofuran containing
10% triethylamine in a Rayonet photochemical reactor (2537.ANG.) by
the method of V. Nair et al., J. Org. Chem., 52, 1344 (1987).
Pharmaceutically acceptable acid salts of compounds 7-18 can be
prepared as described in U.S. Pat. No. 4,383,114, the disclosure of
which is incorporated by reference herein.
The invention will be further described by reference to the
following detailed examples wherein elemental analyses were
performed by M-H-W Laboratories, Phoenix, AZ. Melting points were
determined on a Mel-Temp apparatus and are corrected. Nuclear
magnetic resonance spectra were obtained on Jeol FX 90QFT or
Nicollet NT300 spectrometers and were recorded in DMSO-D.sub.6.
Chemical shifts are expressed in ppm downfield from Me.sub.4 Si. IR
spectra were determined as KBr pellets with a Nicollet 50XC FT-IR
spectrometer, and UV spectra were determined on a Beckmann DU-8
spectrophotometer. Mass spectra were obtained with an AEI
Scientific Apparatus Limited MS-30 mass spectrometer. Thin layer
chromatography (TLC) was performed on 0.25 mm layers of Merck
silica gel 60F-254 and column chromatography on Merck 60 silica gel
(230-400 mesh). All chemicals and solvents are reagent grade unless
otherwise specified.
EXAMPLE 1
(.+-.)-(1.alpha.,4.alpha.)-4-[(5-Amino-6-chloro-4-pyrimidinyl)-amino]-2-cyc
lopentenylcarbinol (3a)
A mixture of 1a (3.0 g, 15 mmol) and aqueous barium hydroxide
(0.5N, 300 ml) was refluxed overnight. After cooling, it was
neutralized with dry ice. The precipitate was filtered out, and the
aqueous solution was concentrated to dryness. The residue was
extracted with absolute ethanol and concentrated again to yield 2a
as a colorless syrup 1.6 g (14 mmol).
To this syrup, 5-amino-4,6-dichloropyrimidine (4.59 g, 28 mmol),
triethylamine (4.2 g, 42 mmol), and n-butanol (50 ml) were added
and the mixture was refluxed for 24 hr. The volatile solvents were
removed, the residue was absorbed on silica gel (7 g), packed in a
flash column (4.0.times.12 cm) and eluted with CHCl.sub.3 -MeOH
(20:1) to yield 2.69 g (74%) of compound 3a; mp
130.degree.-132.degree. C. An analytical sample was obtained by
recrystalization from ethyl acetate (EtOAc), mp
134.degree.-135.degree. C., MS (30 ev, 200.degree. C.); m/e 240 and
242 (M.sup.+ and M.sup.+ +2), 209 (M.sup.+ -31), 144 (B.sup.+); IR:
3600-2600 (OH), 1620,1580 (C.dbd.C, C.dbd.N); Anal. (C.sub.10
H.sub.13 ClN.sub.4 O) C,H,N.
EXAMPLE 2
(.+-.)-(1.alpha.,3.alpha.)-3[(5-Amino-6-chloro-4-pyrimidinyl)-amino]cyclope
ntylcarbinol (3b)
Starting material 1a (2 g, 10 mmol) was dissolved in ethanol (30
ml) and hydrogenated in the presence of 10% palladium-charcoal (20
mg). The catalyst was filtered out and solvent was evaporated. The
residual syrup (1b) was hydrolyzed by a barium hydroxide solution
as described in Example 1, hereinabove.
To the resultant syrup (2b, (10 mmol)),
5-amino-4,6-dichloropyrimidine (20 mmol), triethylamine (30 mmol)
and n-butanol (35 ml) were added and the mixture was refluxed for
24 hr. The reaction mixture was processed as described above for
the synthesis of 3a from 2a to yield 1.62 g (67%) of 3b, mp
161.degree.-163.degree. C. Further recrystalization from ethyl
acetate yielded a material of mp 163.degree.-165.degree. C. MS (30
ev, 200.degree. C.): m/e 242 and 244 (M.sup.+ and M.sup.+ +2), 211
(M.sup.+ -31), 144 (B.sup.+):IR: 3600-2600 (OH), 1620, 1580
(C.dbd.C,C.dbd.N); Anal. (C.sub.10 H.sub.15 ClN.sub.4 O),
C,H,N.
EXAMPLE 3
(.+-.)-(1.alpha.,4.alpha.)-4-[(2-Amino-6-chloro-4-pyrimidinyl)-amino]-2-cyc
lopentenylcarbinol (4a)
To 14 mmol of crude 2a, 2-amino-4,6-dichloropyrimidine (3.74 g,
22.8 mmol), triethylamine (15 ml) and n-butanol (75 ml) were added
and the mixture was refluxed for 48 hr. The volatile solvents were
removed, residue was treated with methanol to separate the
undissolved by-product (the double pyrimidine nucleoside). The
methanol solution was absorbed on silica gel (8 g) packed into a
column (4.0.times.14 cm) and eluted with CHCl.sub.3 -MeOH (40:1) to
yield 1.52 g (42%) of crude 4a. The product was recrystalized from
ethyl acetate to yield 4a; mp 132.degree.-134.degree. C., MS (30
ev, 200.degree. C.); m/e 240 and 242 (M.sup.+ and M.sup.+ +2), 209
(M.sup.+ -31), 144 (B.sup.+); IR: 3600-3000 (NH.sub.2, OH),
1620,1580 (C.dbd.C, C.dbd.N); Anal. (C.sub.10 H.sub.13 ClN.sub.4 O)
C,H,N.
EXAMPLE 4
(.+-.)-(1.alpha.,3.alpha.)-3-[(2-Amino-6-chloro-4-pyrimidinyl)-amino]cyclop
entylcarbinol (4b)
To 2b (25 mmol), 2-amino-4,6-dichloropyrimidine (37.5 mmol),
triethylamine (25 ml) and n-butanol (125 ml) were added and the
mixture was refluxed for 48 hr. It was processed as described in
Example 4a to yield 2.7 g (44%) of crystaline 4b, mp
122.degree.-124.degree. C. Recrystalization from ethyl acetate
yielded 4b of mp 124.degree.-126.degree. C. MS (30 ev, 200.degree.
C.); m/e 242 and 244 (M.sup.+ and M.sup.+ +2), 211 (M.sup.+ -31),
144 (B.sup.+); IR: 3600-3000 (NH.sub.2, OH), 1620,1580 (C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.15 ClN.sub.4 O) C,H,N.
EXAMPLE 5
(.+-.)-(1.alpha.,4.alpha.)-4-{[(2-Amino-6-chloro-5-(4-chlorophenyl)azo]-4-p
yrimidinyl-amino}-2-cyclopentenylcarbinol (5a)
A cold diazonium salt solution was prepared from p-chloroaniline
(1.47 g, 11.5 mmol) in 3N HCl (25 ml) and sodium nitrite (870 mg,
12.5 mmol) in water (10 ml). This solution was added to a mixture
of 4a (2.40 g, 10 mmol), acetic acid (50 ml), water (50 ml) and
sodium acetate trihydrate (20 g). The reaction mixture was stirred
overnight at room temperature. The yellow precipitate was filtered
and washed with cold water until neutral, then it was air-dried in
the fumehood to yield 3.60 g (94%), of 5a, mp 229.degree. C. (dec).
The analytical sample was obtained from acetone-methanol (1:2), mp
241.degree.-243.degree. C. (dec). MS (30 ev, 260.degree. C.): m/e
378 and 380 (M.sup.+ and M.sup.+ +2), 282 (B.sup.+); IR: 3600-3000
(NH.sub.2, OH), 1620,1580 (C.dbd.C, C.dbd.N); Anal. (C.sub.16
H.sub.16 Cl.sub.2 N.sub.6 O) C,H,N.
EXAMPLE 6
(.+-.)-(1.alpha.,3.alpha.)-3-{[(2-Amino-6-chloro-5-(4-chlorophenyl)azo]-4-p
yrimidinyl-amino}-cyclopentylcarbinol (5b).
A cold diazonium salt solution prepared as described in Example 5
was added to a mixture of 4b (2.42 g, 10 mmol), acetic acid (50 ml)
water (50 ml) and sodium acetate trihydrate (20 g). Following the
procedures of Example 5, compound 5b was obtained as a yellow
product, 3.69 g (94%), mp 260.degree.-262.degree. C. (dec). The
crude product was recrystalized from acetone-methanol (3:1), MS (30
ev, 260.degree. C.); m/e 380 and 382 (M.sup.+ and M.sup.+ +2), 282
(B.sup.+); IR: 3600-3000 (NH.sub.2, OH), 1620,1580 (C.dbd.C,
C.dbd.N); Anal. (C.sub.16 H.sub.18 Cl.sub.2 N.sub.6 O) C,H,N.
EXAMPLE 7
(.+-.)-(1.alpha.,4.alpha.)-4-[(2,5-Diamino-6-chloro-4-pyrimidinyl-amino]-2-
cyclopentenylcarbinol (6a).
A mixture of 5a (379 mg, 1 mmol), zinc dust (0.65 g, 10 mmol),
acetic acid (0.32 ml), water (15 ml) and ethanol (15 ml) was
refluxed under nitrogen for 3 hr. The zinc was removed and the
solvents were evaporated. The residue was absorbed on silica gel (2
g), packed into a column (2.0.times.18 cm), and eluted with
CHCl.sub.3 -MeOH (15:1). A pink syrup was obtained. Further
purification from methanol-ether yielded 6a as pink crystals, 170
mg (66%), mp 168.degree.-170.degree. C., MS (30 ev, 220.degree.
C.); m/e 255 and 257 (M.sup.+ and M.sup.+ +2), 224 (M.sup.+ -31),
159 (B.sup.+); IR: 3600-3000 (NH.sub.2, OH), 1620,1580 (C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.14 ClN.sub.5 O) C,H,N.
EXAMPLE 8
(.+-.)-(1.alpha.,3.alpha.)-3-[(2,5-Diamino-6-chloro-4-pyrimidinyl)amino]-cy
clopentylcarbinol (6b)
A mixture of 5b (3.08 g, 8.1 mmol), zinc dust (5.2 g, 80 mmol),
acetic acid (2.6 ml), water (130 ml) and ethanol (130 ml) was
refluxed under nitrogen for 3 hr, and worked up as described in
Example 7, above. The mixture was absorbed on silica gel (18 g),
packed in a column (4.0.times.8 cm) and eluted with CHCl.sub.3
-MeOH (20:1) to yield 6b as yellow-pink crystals, 1.44 g (69%). The
product was recrystalized from methanolether to yield 6b, mp
143.degree.-145.degree. C., MS (30 ev, 200.degree. C.); m/e 257 and
259 (M.sup.+ and M.sup.+ +2), 226 (M.sup.+ -31), 159 (B.sup.+); IR:
3600-3000 (NH.sub.2, OH), 1620,1580 (C.dbd.C, C.dbd.N); Anal.
(C.sub.10 H.sub.16 ClN.sub.5 O) C,H,N.
EXAMPLE 9
(.+-.)-(1.alpha.,4.alpha.)-4-(6-chloro-9H-purin-9-yl)-2-cyclopentenylcarbin
ol (7a)
A mixture of 3a (1.30 g, 5.4 mmol), triethyl orthoformate (30 ml)
and hydrochloric acid (12N, 0.50 ml) was stirred overnight at room
temperature. The solvent was evaporated at 35.degree. C. in vacuo.
To the residue was added aqueous hydrochloric acid (0.5N, 30 ml)
and the mixture was stirred for 1 hr. The mixture was neutralized
to pH 7-8 with 1N sodium hydroxide and absorbed onto silica gel (8
g), packed in a column (4.0.times.8 cm), and eluted with CHCl.sub.3
-MeOH (20:1) to yield white crystals of 7a, 1.12 g (82%). The crude
product was recrystalized from ethyl acetate to yield 7a, mp
108.degree.-110.degree. C., MS (30 ev, 200.degree. C.); m/e 250 and
252 (M.sup.+ and M.sup.+ +2), 219 (M.sup.+ -31), 154 (B.sup.+); IR;
3600-2800 (OH), 1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.11
ClN.sub.4 O) C,H,N.
EXAMPLE 10
(.+-.)-(1.alpha.,3.alpha.)-3-(6-Chloro-9H-purin-9-yl)-cyclopentylcarbinol
(7b)
A mixture of 3b (1.29 g, 5.3 mmol), triethyl orthoformate (30 ml)
and hydrochloric acid (12N, 0.50 ml) was stirred overnight at room
temperature and processed as described in Example 9 to yield white
crystals of 7b, 1.15 g (86%). The crude product was recrystalized
from ethyl acetate to yield 7b, mp 120.degree.-122.degree. C., MS
(30 ev, 180.degree. C.); m/e 252 and 254 (M.sup.+ and M.sup.+ +2),
221 (M.sup.+ -31), 154 (B.sup.+); IR; 3600-2800 (OH), 1600
(C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.13 ClN.sub.4 O)
C,H,N.
EXAMPLE 11
(.+-.)-(1.alpha.,4.alpha.)-4-(6-Hydroxy-9H-purin-9-yl)-2-cyclopentenyl-carb
inol (8a)
A mixture of 7a (251 mg, 1 mmol) and aqueous sodium hydroxide
(0.2N, 10 ml) was refluxed for 3 hr. After cooling, the reaction
mixture was adjusted to pH 5-6 with acetic acid. The reaction
mixture was absorbed on silica gel (2 g) packed in a column
(2.0.times.11 cm) and eluted with CHCl.sub.3 -MeOH (10:1) to yield
105 mg (45%) of 8a. The crude white product was recrystalized from
water-methanol (3:1) to yield 8a, mp 248.degree.-250.degree. C.
(dec), MS (30 ev, 300.degree. C.); m/e 232 (M.sup.+), 214 (M.sup.+
-18), 136 (B.sup.+); IR; 3600-2600 (OH), 1680,1600 (C.dbd.O,
C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.12 N.sub.4 O.sub.2)
C,H,N.
EXAMPLE 12
(.+-.)-(1.alpha.,3.alpha.)-3-(6-Hydroxy-9H-purin-9-yl)-cyclopentylcarbinol
(8b)
A mixture of 7b (378 mg, 1.5 mmol) and aqueous sodium hydroxide
(0.2N, 15 ml) was refluxed for 3 hr. The mixture was processed as
described in Example 11 to yield 213 mg (60%) of 8b, mp
276.degree.-278.degree. C. (dec), MS (30 ev, 280.degree. C.); m/e
234 (M.sup.+), 216 (M.sup.+ -18), 136 (B.sup.+); IR: 3600-2600
(OH), 1680,1600 (C.dbd.O, C.dbd.C, C.dbd.N); Anal. (C.sub.11
H.sub.14 N.sub.4 O.sub.2) C,H,N.
EXAMPLE 13
(.+-.)-(1.alpha.,4.alpha.)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentenylcarbino
l (9a)
Liquid ammonia was passed into a bomb containing a solution of 7a
(250 mg, 1 mmol) in methanol (5 ml) at -80.degree. C. The bomb was
sealed and heated at 60.degree. C. for 24 hr. Ammonia and methanol
were evaporated and the residue was recrystalized from water to
yield off-white crystals of 9a, 187 mg (81%), mp
198.degree.-200.degree. C. MS (30 ev, 210.degree. C.): m/e 231
(M.sup.+), 213 (M.sup.+ -18), 135 (B.sup.+); IR: 3600-2600
(NH.sub.2, OH), 1700,1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.11
H.sub.13 N.sub.5 O) C,H,N.
EXAMPLE 14
(.+-.)-(1.alpha.,3.alpha.)-3-(6-Amino-9H-purin-9-yl)-cyclopentylcarbinol
(9b).
Compound 7b (252 mg, 1 mmol) was reacted with ammonia and processed
as described in Example 13. The residue was absorbed on silica gel
(0.8 g) packed in a column (1.0.times.10 cm) and eluted with
CHCl.sub.3 -MeOH (10:1). The resultant product was recrystalized
from methanolethyl acetate to yield white crystals of 9b, 179 mg
(77%), mp 146.degree.-148.degree. C. MS (70 ev, 200.degree. C.):
m/e 233 (M.sup.+), 216 (M.sup.+ -17), 135 (B.sup.+); IR: 3600-2600
(NH.sub.2, OH), 1700,1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.11
H.sub.15 N.sub.5 O) C,H,N.
EXAMPLE 15
(.+-.)-(1.alpha.,4.alpha.)-4-(6-Mercapto-9H-purin-9-yl)-2-cyclopentenyl-car
binol (10a)
A mixture of 7a (125 mg, 0.5 mmol), thiourea (40 mg, 0.64 mmol) and
n-propanol (5 ml) was refluxed for 2 hr. After cooling, the
precipitate was isolated by filtration, washed with n-propanol, and
dissolved in sodium hydroxide (1N, 5 ml). The solution was adjusted
to pH 5 with acetic acid. The crude 10a (90 mg, 73%) was isolated
again, mp 260.degree.-262.degree. C. (dec) and was recrystalized
from N,N-dimethylformamide, to yield 10a, mp
263.degree.-265.degree. C. (dec). MS (30 ev, 290.degree. C.): m/e
248 (M.sup.+), 230 (M.sup.+ -18), 152 (B.sup.+); IR: 3600-3200
(OH), 3100,2400 (SH), 1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.11
H.sub.12 N.sub.4 OS) C,H,N.
EXAMPLE 16
(.+-.)-(1.alpha.,3.alpha.)-3-(6-Mercapto-9H-purin-9-yl)-cyclopentylcarbinol
(10b )
A mixture of 7b (252 mg, 1 mmol), thiourea (95 ml, 1.25 mmol) and
n-propanol (12 ml) was refluxed for 2 hr. The reaction mixture was
processed as described in Example 15 to yield a crude product which
was recrystalized from methanol to yield white crystals of 10b, 173
mg (69%), mp 297.degree.-300.degree. C. (dec). MS (30 ev,
280.degree. C.): m/e 250 (M.sup.+), 233 (M.sup.+ -17), 152
(B.sup.+); IR: 3600-3200 (OH), 3100,2400 (SH), 1600 (C.dbd.C,
C.dbd.N); Anal. (C.sub.11 H.sub.14 N.sub.4 OS) C,H,N.
EXAMPLE 17
(.+-.)-(1.alpha.,4.alpha.)-4-(7-Hydroxy-3H-1,2,3-triazolo[4,5-d]pyrimidin-3
-yl)-2-cyclopentenyl carbinol (11a).
To a cold solution of 3a (361 mg, 1.5 mmol) in hydrochloric acid
(1N, 30 ml) was added sodium nitrite solution (120 mg, 1.7 mmol) in
3 ml of water. The reaction was monitored by starch-potassium
iodide paper. The mixture was concentrated at 40.degree. C. to a
volume of 2 ml and adjusted to pH 7 with aqueous sodium hydroxide.
The mixture was absorbed on silica gel (2 g), packed in a column
(2.0.times.13 cm) and eluted with CHCl.sub.3 -MeOH (10:1). The
crude 11a was recrystallized from water-methanol (3:1) to yield
white crystals of 11a, 173 mg (49%) mp 180.degree.-182.degree. C.
MS (30 ev, 230.degree. C.): m/e 233 (M.sup.+), 203 (M.sup.+ -30),
137 (B.sup.+); IR: 3600-2600 (OH), 1740,1600 (C.dbd.O, C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.11 N.sub.5 O.sub.2) C,H,N.
EXAMPLE 18
(.+-.)-(1.alpha.,3.alpha.)-3-(7-Hydroxy-3H-1,2,3-triazolo[4,5-d]pyrimidin-3
-yl)-cyclopentyl carbinol (11b)
Compound 3b (363 mg, 1.5 mmol) was processed as described in
Example 17 and recrystallized from water-methanol to yield 242 mg
(269%) of 11b, mp 179.degree.-182.degree. C. MS (30 ev, 280.degree.
C.): m/e 235 (M.sup.+), 204 (M.sup.+ -31), 137 (B.sup.+); IR:
3600-2600 (OH), 1740,1600 (C.dbd.O, C.dbd.C, C.dbd.N); Anal.
(C.sub.10 H.sub.13 N.sub.5 O.sub.2) C,H,N.
EXAMPLE 19
(.+-.)-(1.alpha.,4.alpha.)-4-(7-Amino-3H-1,23,-triazolo[4,5-d]pyrimidin-3-y
l)-2-cyclopentenyl carbinol (12a)
Sodium nitrite solution (828 mg, 12 mmol) in water (10 ml) was
added dropwise to a cold solution of 3a (2.43 g, 10.1 mmol) in
hydrochloric acid (0.5N, 40 ml). The reaction mixture was stirred
at room temperature for 1 hr, then concentrated to a syrup. The
syrup was dissolved in ethanol and transferred into a strainless
steel bomb. Liquid ammonia was passed in, the bomb was sealed, and
the reaction mixture was stirred at room temperature overnight.
Ammonia was evaporated and the residue was chromatographed on
silica gel (150 g) eluting with CH.sub.2 CL.sub.2 -MeOH (10:1) to
yield white crystals of 12a, 1.62 g (69%), mp
220.degree.-222.degree. C. (dec). MS (30 ev, 220.degree. C.): m/e
232 (M.sup.+), 202 (M.sup.+ -30), 136 (B.sup.+); IR: 3600-2800
(NH.sub.2, OH), 1700,1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.10
H.sub.12 N.sub.6 O) C,H,N.
EXAMPLE 20
(.+-.)-(1.alpha.,3.alpha.)-3-(7-Amino-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-y
l)-cyclopentyl carbinol (12b)
Compound 12a (200 mg, 0.86 mmol) was dissolved in ethanol (25 ml)
and hydrogenated in the presence of 10% palladium-charcoal (30 mg)
under 49 lbs/in.sup.2 of H.sub.2 for 18 hrs. The catalyst was
removed and the solvent was evaporated. The resultant crude product
was recrystallized from ethanol-water (1:2) to yield white crystals
of 12b, 173 mg (86%), mp 195.degree.-196.degree. C. MS (70 ev,
190.degree. C.): m/e 234 (M.sup.+), 203 (M.sup.+ -31), 136
(B.sup.+); IR: 3600-2800 (NH.sub.2, OH), 1700,1600 (C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.14 N.sub.6 O) C,H,N.
EXAMPLE 21
(.+-.)-(1.alpha.,4.alpha.)-4-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopenten
yl carbinol (13a)
A mixture of 6a (1.41 g, 5.5 mmol) treithyl orthoformate (30 ml)
and hydrochloric acid (12N, 140 ml) was stirred overnight. The
suspension was dried in vacuo. Diluted hydrochloric acid (0.5N, 40
ml) was added and the mixture was reacted at room temperature for 1
hr. The mixture was neutralized to pH 8 with 1N sodium hydroxide
and absorbed on silica gel (7.5 g) packed in a column (4.0.times.10
cm) and eluted by CHCl.sub.3 -MeOH (20:1) to yield off-white
crystals of 13a, 118 g (80%). The crude product was recrystalized
from ethanol to yield 13a, mp 145.degree.-147.degree. C. MS (30 ev,
220.degree. C.): m/e 265 and 267 (M.sup.+ and M.sup.+ +2), 235
(M.sup.+ -30), 169 (B.sup.+); IR: 3600-2600 (NH.sub.2, OH),
1620,1580 (C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.12 N.sub.5
OCl.multidot.3/4 H.sub.2 O) C,H,N.
EXAMPLE 22
(.+-.)-(1.alpha.,3.alpha.)-3-(2-Amino-6-chloro-9H-purin-9-yl)-cyclopentyl
carbinol (13b)
A mixture of 6b (1.3 g, 5 mmol) triethyl orthoformate (30 ml) and
hydrochloric acid (12N, 1.30 ml) was stirred overnight. The
reaction mixture was processed as described in Example 21 to yield
off-white crystals of 13b, 1.00 g (73%), mp 151.degree.-153.degree.
C. MS (30 ev, 230.degree. C.): m/e 267 and 269 (M.sup.+ and M.sup.+
+2), 236 (M.sup.+ -31), 169 (B.sup.+); IR: 3600-2600 (NH.sub.2,
OH), 1620,1580 (C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.14 N.sub.5
OCl.multidot.1/2 H.sub.2 O) C,H,N.
EXAMPLE 23
(.+-.)-(1.alpha.,4.alpha.)-4-(2-Amino-6-hydroxy-9H-purin-9-yl)-2-cyclopente
nyl carbinol (14a)
A mixture of 13a (266 mg, 1 mmol) and aqueous sodium hydroxide
(0.33N) was refluxed for 5 hr, absorbed onto silica gel (2 g)
packed in a column (2.0.times.7.5 cm) and eluted with CHCl.sub.3
-MeOH (5:1). The crude product was recrystalized from
methanol-water (1:4) to yield white crystals of 14a, 152 mg (61%),
mp 254.degree.-256.degree. C. (dec). MS (30 ev, 200.degree. C.):
m/e 247 (M.sup.+), 127 (M.sup.+ -30), 151 (B.sup.+); IR: 3600-2600
(NH.sub.2, OH), 1700,1600 (C.dbd.O, C.dbd.C, C.dbd.N); Anal.
(C.sub.11 H.sub.13 N.sub.5 O.sub.2 .multidot.3/4 H.sub.2 O)
C,H,N.
EXAMPLE 24
(.+-.)-(1.alpha.,3.alpha.)-3-(2-Amino-6-hydroxy-9H-purin-9-yl)-cyclopentyl
carbinol (14b)
Compound 13b (280 mg, 1 mmol) was hydrolyzed with sodium hydroxide
and purified as described in Example 23 to yield white crystals of
14b, 204 mg (77%), mp 274.degree.-276.degree. C. (dec). MS (30 ev,
320.degree. C.): m/e 249 (M.sup.+), 218 (M.sup.+ -31), 151
(B.sup.+); IR: 3600-2600 (NH.sub.2, OH), 1700,1600 (C.dbd.O,
C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.15 N.sub.5 O.sub.2
.multidot.3/4 H.sub.2 O) C,H,N.
EXAMPLE 25
(.+-.)-(1.alpha.,4.alpha.)-4-(2,6-Diamino-9H-purin-9-yl)-2-cyclopentenyl
carbinol (15a)
Liquid ammonia was passed into a solution of 13a (265 mg, 1 mmol)
in methanol (10 ml) at -80.degree. C. in a bomb. The bomb was
sealed and heated at 75.degree. C. for 48 hr. Ammonia and methanol
were evaporated. The residue was absorbed on silica gel (2 g),
packed in a column (2.0.times.10 cm) and eluted with CHCl.sub.3
-MeOH (15:1). The crude product was recrystalized from ethanol to
yield 196 mg (80%) of 15a, mp 152.degree.-155.degree. C. MS (30 ev,
200.degree. C.): m/e 246 (M.sup.+), 229 (M.sup.+ -17), 216 (M.sup.+
-30), 150 (B.sup.+); IR: 3600-3000 (NH.sub.2, OH), 1700,1650,1600
(C.dbd.O, C.dbd.C, C.dbd.N); Anal. (C.sub.11 H.sub.14 N.sub.6 O)
C,H,N.
EXAMPLE 26
(.+-.)-(1.alpha.,3.alpha.)-3-(2,6-Diamino-9H-purin-9-yl)-cyclopentyl
carbinol (15b)
Compound 13b (280 mg, 1 mmol) reacted with ammonia as described in
Example 25 to yield white crystals of 15b, 193 mg (77%), mp
208.degree.-211.degree. C. MS (30 ev, 220.degree. C.): m/e 248
(M.sup.+), 231 (M.sup.+ -17), 217 (M.sup.+ -31), 150 (B.sup.+); IR:
3600-3000 (NH.sub.2, OH), 1700,1650,1600 (C.dbd.O, C.dbd.C,
C.dbd.N); Anal. (C.sub.11 H.sub.16 H.sub.6 O) C,H, N.
EXAMPLE 27
(35
)-(1.alpha.,4.alpha.)-4-(5-Amino-7-chloro-3H-1,2,3-triazolo[4,5-d]pyrimidi
n-3-yl)-2-cyclopentenyl carbinol (16a)
To a cold solution of 6a (255 mg, 1 mmol) in acetic acid (1.5 ml)
and water (2.5 ml) was added sodium nitrite (83 mg, 1.2 mmol) in
water (2 ml). The reaction was monitored by starch-potassium iodide
paper. After stirring for 1 hr at 0.degree. C., the precipitate was
filtered and washed with cold water, then dried over phosphorus
pentoxide in vacuo to yield 16a as off-white crystals, 218 mg
(81%). The crude 16a was recrystalized from methanol, mp
153.degree.-155.degree. C. (dec). MS (30 ev, 220.degree. C.): m/e
266 and 268 (M.sup.+ and M.sup.+ +2), 236 (M.sup.+ -30), 170
(B.sup.+); IR: 3600-3000 (NH.sub.2, OH), 1650,1600 (C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.11 ClN.sub.6 O) C,H,N.
EXAMPLE 28
(.+-.)-(1.alpha.,3.alpha.)-3-(5-Amino-7-chloro-3H-1,2,3-triazolo[4,5-d]pyri
midin-3-yl)-cyclopentyl carbinol (16)
Compound 6b (773 mg, 3 mmol) was reacted with sodium nitrite as
described in Example 27 to yield 666 mg (82%) of 16b, which was
recrystalized from methanol, mp 170.degree.-172.degree. C. (dec).
MS (20 ev, 300.degree. C.): m/e 268 and 270 (M.sup.+ and M.sup.+
+2), 237 (M.sup.+ -31), 170 (B.sup.+); IR: 3600-3000 (NH.sub.2,
OH), 1650,1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.10 H.sub.13
ClN.sub.6 O) C,H,N.
EXAMPLE 29
(.+-.)-(1.alpha.,4.alpha.),-4-(5-Amino-7-hydroxy-3H-1,2,3-triazolo[4,5-d]py
rimidin-3-yl)-2-cyclopentenyl carbinol (17a)
A mixture of 16a (218 mg, 0.8 mmol) and aqueous sodium hydroxide
(0.25N, 10 ml) was refluxed for 3 hr, then was adjusted to pH 3
with 6N hydrochloric acid. The gelatinious precipitate was filtered
and washed with cold water. It was dried over phosphorous pentoxide
in vacuo to yield 17a as an off-white solid, 181 mg (90%) mp
222.degree.-224.degree. C. (dec). After recrystalization from
water, the mp was 223.degree.-225.degree. C. (dec). MS (20 ev,
300.degree. C.): m/e 248 (M.sup.+), 217 (M.sup.+ -31), 152
(B.sup.+); IR: 3600-3000 (NH.sub.2, OH), 1750,1600 (C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.12 N.sub.6 O.sub.2 .multidot.1/2
H.sub.2 O) C,H,N.
EXAMPLE 30
(.+-.)-(1.alpha.,3.alpha.)-3-(5-Amino-7-hydroxy-3H-1,2,3-triazolo[4,5-d]pyr
imidin-3-yl)-cyclopentyl carbinol (17b)
Compound 16b (268 mg, 1 mmol) was hydrolyzed and processed as
described in Example 29 to yield 17b as an off-white product, 203
mg (81%). The crude product was recrystalized from water to yield
17b, mp 228.degree.-231.degree. C. MS (20 ev, 320.degree. C.): m/e
250 (M.sup.+), 219 (M.sup.+ -31), 152 (B.sup.+); IR: 3600-3000
NH.sub.2, OH), 1750,1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.10
H.sub.14 N.sub.6 O.sub.2 .multidot.1.25 H.sub.2 O) C,H,N.
EXAMPLE 31
(.+-.)-(1.alpha.,4.alpha.)-4-(5,7-Diamino-3H-1,2,3-triazolo[4,5-]pyrimidin-
3-yl)-2-cyclopentenyl carbinol (18a)
Compound 16a (267 mg, 1 mmol) was processes as described in Example
25, employing a reaction time of 60.degree. C. for 20 hr. The
residual mixture was absorbed on silica gel (2 g), packed in a
column (2.0.times.10 cm) and eluted by CHCl.sub.3 -MeOH (15:1) to
yield 18a as white crystals, 204 mg (83%). The crude product was
recrystalized from ethanol-water (2:1), to yield 18a of mp
240.degree.-242.degree. C. (dec). MS (30 ev, 240.degree. C.): m/e
247 (M.sup.+), 229 (M.sup.+ -18), 217 (M.sup.+ -30), 151 (B.sup.+);
IR: 3600-3100 (NH.sub.2, OH), 1700,1650,1600 (C.dbd.O, C.dbd.C,
C.dbd.N); Anal. (C.sub.10 H.sub.13 N.sub.7 O.multidot.H.sub.2 O)
C,H,N.
EXAMPLE 32
(.+-.)-(1.alpha.,3.alpha.)-3-(5,7-Diamino-3H-
1,2,3-triazolo[4,5-d]pyrimidin-3-yl)-cyclopentyl carbinol (18b)
Compound 16b (268 mg, 1 mmol) was processed as described in Example
31 to yield 220 mg of 18b (88%), which was recrystalized from
ethanol-water (1:2) to afford pink-white crystals, mp
223.degree.-225.degree. C. MS (30 ev, 250.degree. C.): m/e 249
(M.sup.+), 218 (M.sup.+ -31), 151 (B.sup.+); IR: 3600-3100
(NH.sub.2, OH), 1700,1600 (C.dbd.C, C.dbd.N); Anal. (C.sub.10
H.sub.15 N.sub.7 O) C,H, N.
EXAMPLE 33
(.+-.)-(1.alpha.,4.alpha.)-4-(3-Methoxy-2-methylacryloylureido)-2-cyclopent
enyl carbinol (19a)
Isocyanate reagent was prepared from 3-methoxy-2-methylacryloyl
chloride (1.00 g, bp 65.degree.-66.degree. C./2.5 mm) in anhydrous
benzene (10 ml) and freshly dried silver cyanate (2.6 g, 17 mmol,
dried at 110.degree. C., 2 hr) by refluxing for 0.5 hr. The
supernatant was added dropwise into a solution of 2a (from 1a, 0.8
g, 4 mmol) in N,N-dimethylformamide (10 ml) at -15.degree. C. and
the mixture was stirred for 1 hr, then stored at 4.degree. C.
overnight. The solvent was evaporated and the residue was absorbed
on silica gel (3 g), packed in a column (2.0.times.16 cm) and
eluted with CHCl.sub.3 -MeOH (20:1) to yield white crystals of 19a,
605 mg, (60%), mp 147.degree.-149.degree. C. MS (30 ev, 200.degree.
C.): m/e 254 (M.sup.+), 239 (M.sup.+ -15), 223 (M.sup.+ -31), 158
(B.sup.+); IR: 3600-2800 (NH.sub.2, OH), 1700,1650,1600 (C.dbd.O,
C.dbd.C); Anal. (C.sub.12 H.sub.18 N.sub.2 O.sub.4) C,H,N.
EXAMPLE 34
(.+-.)-(1.alpha.,4.alpha.)-4-[5-Methyl-2,4-(1H,3H)-pyrimidinedion-3-yl]-2-c
yclopentenyl carbinol (20a)
A mixture of 19a (381 mg, 1.5 mmol), p-toluenesuflonic acid
monohydrate (20 mg) and anhydrous N,N-dimethylformamide (2 ml) was
stirred at 115.degree. C. for 3 hr. The solvent was evaporated, the
residue was absorbed on silica gel (3 g), packed in a column
(2.0.times.14 cm) and eluted with CHCl.sub.3 -MeOH (20:1) to yield
20a as off-white crystals, 206 mg (62%). The product was
recrystalized from absolute ethanol to yield 20a, mp
213.degree.-215.degree. C. MS (30 ev, 250.degree. C.): m/e 222
(M.sup.+), 204 (M.sup.+ -18), 191 (M.sup.+ -31), 126 (B.sup.+); IR:
3600-3300 (OH), 1700,1600 (C.dbd.O, C.dbd.C); Anal. (C.sub.11
H.sub.14 N.sub.2 O.sub.3) C,H,N.
EXAMPLE 35
Cytotoxicity Assay
The ED.sub.50 cytotoxicity concentration determined for analogs
16a, 16b, 17a and 17b in the P-388 mouse leukemia cell culture
assay are given in Table II.
TABLE II ______________________________________ Inhibitory
Concentrations of Carbocyclic Nucleosides for P-388 Leukemia Cells
in Cultures* Compound ED.sub.50, mg/ml
______________________________________ 16a 1.0 16b 12.5 17a 4.5 17b
50.0 ______________________________________ *Assay Technique: R. G.
Almquist and R. Vince, J. Med. Chem., 16, 1396 (1973).
EXAMPLE 36
Anti-HIV Assay
Compound 14a was screened for anti-HIV activity at the National
Cancer Institute, Frederick Cancer Research Facility, Frederick,
Md. (FCRF). The following are the current screening mode
operational procedures utilized at FCRF. The protocol consists of 3
areas, (I) preparation of infected cells and distribution to the
test plates, (II) preparation of drug dilution plates and
distribution to the test plates, and (III) XTT assay procedure. See
D. A. Scudiero et al., "A New Simplified Tetrazolium Assay for Cell
Growth and Drug Sensitivity in Culture," Cancer Res., 48,4827
(1988).
I. Infection and Distribvution of ATH8 Cells to Microtiter
Trays
The AYTH8 cells (a normal lymphoblastoid cell line which expresses
CD4) are placed in 50 ml conical centrifuge tubes and treated for 1
hr with 1-2 .mu.g/ml of polybrene at 37.degree. C. The cells are
then pelleted for 8 min. at 1200 RPM. HIV virus, diluted 1:10 in
media (RMP1-1640, 10% human serum or 15% fetal calf serum (FSC),
with IL-2 and antibiotics) is added to provide an MOI of 0.001.
Medium alone is added to virus-free control cells. Assuming an
infectious virus titer of 10.sup.-4, an MOI of 0.001 represents 8
infectious virus particles per 10,000 cells. About 500,000
cells/tube are exposed to 400 .mu.l of the virus dilution. The
resultant mixture is incubated for 1 hr at 37.degree. C. in
Air-CO.sub.2. The infected or uninfected cells are diluted to give
1.times.10.sup.-4 (with human serum or 2.times.10.sup.-4 (with
fetal calf serum) cells/100 .mu.l.
Infected or uninfected cells (100 .mu.l) are distributed to
appropriate wells of a 96 well, U-bottom, microtiter plate. Each
compound dilution is tested in duplicate with infected cells.
Uninfected cells are examined for drug sensitivity in a single well
for each dilution of compound. Drug-free control cells, infected
and unifected, are run in triplicate. Wells B2 through G2 served as
reagent controls and received medium only. The plates are incubated
at 37.degree. C. in Air-CO.sub.2 until the drug is added.
II. Drug Dilution and Addition
Dilution plates (flat bottom 96 well, microtiter plates) are
treated overnight with phosphate buffered saline (PBS) or media
containing at least 1% FCS or 1% human serum (depending on the
medium used in the test), beginning the day before assay. This
"blocking" procedure is used to limit the absorption of drug to the
mcirotiter tray during the dilution process. The wells are filled
completely with the blocking solution and allowed to stand at room
temperature in a humidified chamber in a hood.
The dilution process is begun by first diluting the test compound
1:20. Blocked, dilution plates are prepared by flicking out the
blocking solution and blotting dry on sterile gauze. All wells of
each plate are then filled with 225 .mu.l of the appropriate medium
using a Cetus liquid handling system. Twenty-five microliters (25
.mu.l) of each 1:20 diluted compound is then manually added to row
A of a blocked and filled dilution plate. Four compounds,
sufficient to supply two test plates, are added per dilution plate.
The four compounds are then serially diluted 10 fold from row A
through row H using the Cetus liquid handling system. The starting
dilution of each compound in row A is, at this point, 1:200. The
dilution plates are kept on ice until needed.
Using a multi-channel pipettor with 6 microtips, 100 .mu.l of each
drug dilution is transferred to the test plate which already
contains 100 .mu.l of medium plus cells. The final dilution, in the
test plate, starts at 1:400 (wells B4 through G4). This dilution
(to 0.25% DMSO) prevents the DMSO vehicle from interfering with
cell growth. Drug-free, infected or uninfected cells (wells B3
through G3) and reagent controls (B2 through G2) receive medium
alone. The final 2 compounds are then transferred from wells H7
through H12 to a second test plate using the same procedure. Test
plates are incubated at 37.degree. C. in Air-CO.sub.2 for 7-14 days
or until virus controls are lysed as determined
macroscopically.
III. Quantitation of Viral Cytopathogenicity and Drug Activity
A. Materials
1. A solution of
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)
carbonyl]-2-H-tetrazolium hydroxide. (XTT) - 1 mg/ml sulution in
media without FCS. Store at 4.degree. C. Prepare weekly.
2. Phenazine methosulfonate (PMS) stock solution - This Can be
prepared and maintained frozen until needed at -20.degree. C. It
should be made in PBS to a concentration of 15.3 mg/ml.
B. Microculture Tetrazolium Assay (MTA)
1. Preparation of XTT-PMS Solution - The XTT-PMS is prepared
immediately prior to its addition to the wells of the culture dish.
The stock PMS solution is diluted 1:100 (0.153 mg/ml). Diluted PMS
is added to every ml of XTT required to give a final PMS
concentration of 0.02 mM. A 50 .mu.l aliquot of the XTT-PMS mixture
is added to each of the appropriate wells, and the plate is
incubated for four hours at 37.degree. C. The plate lids are
removed and replaced with adhesive plate sealers (Dynatech cat
001-010-3501). The sealed plate is shaken on a microculture plate
mixer and the absorbance is determined at 450 nm.
IV. Results
FIG. 2 depicts a plot of the percentage of test cells over
uninfected cells (%) for both infected and uninfected cells as a
function of the increasing concentration of compound 14a.
The data plotted on FIG. 2 permit the calculation of an effective
concentration (EC.sub.50) with respect to infected cells of about
0.15 .mu.g/ml, an inhibitory concentration (IC.sub.50) with respect
to normal cells of about 100 .mu.g/ml, and a therapeuitc index
(TI.sub.50) of about 667. An earlier assay carried out at the
Southern Research Institute yielded at TI.sub.50 of about 200 when
MT-2 cells were cultured with H9/HTLV-IIIB.
The invention comprises the biologically active compounds as
disclosed or the pharmaceutically acceptable salts or esters
thereof, together with a pharmaceutically acceptable carrier for
administration in effective non-toxic dose form. Pharmaceutically
acceptable salts may be salts of organic acids, such as acetic,
lactic, malic or p-toluene sulphonic acid and the like as well as
salts of pharmaceutically acceptable mineral acids, such as
hydrochloric or sulfuric acid and the like. Other salts may be
prepared and then converted by conventional double decomposition
methods into pharmaceutically acceptable salts directly suitable
for purposes of treatment of viral infections in mammals or for the
prevention of viral contamination of physiological fluids such as
blood or semen in vitro.
The compounds of the general formula I or II may exist in the form
of optical isomers, and these isomers, as well as racemic (.+-.)
mixtures are included within the compounds of this invention.
Pharmaceuticaloly acceptable carriers are materials useful for the
purpose of administering the present analogs and may be solid,
liquid or gaseous materials, which are otherwise inert and
medically acceptable and are compatible with the active
ingredients. Thus, the present active compounds can be combined
with the carrier and added to physiological fluids in vitro or
administered in vivo parenterally, orally, used as a suppository or
pessary, applied topically as an ointment, cream, acrosol, powder,
or given as eye or nose drops, etc., depending upon whether the
prepartion is used for treatment of internal or external viral
infections.
For internal virus infections, the compositions may be administered
orally or parenterally at effective non-toxic antivirus dose levels
of about 10 to 750 mg/kg/day of body weight given in one dose or
several smaller doses throughout the day. For oral administration,
fine powders or granules may contain diluting, dispersing and/or
surface active agents and may be presented in water or in a syrup;
in capsules in the dry state, or in a non-aqueous solution or
suspension; in tablets or the like. Where desirable or necessary,
flavoring, preserving, suspending, thickening or emulsifying agents
may be included. For parenteral administration, administration as
drops, the compounds may be presented in aqueous solution in an
effective non-toxic dose in concentration of from about 0.1 to 10
percent w/v. The solutions may contain antoxidants, buffers and the
like. Alternatively, for infections of external tissues, the
compositions are preferably applied as a topical ointment or cream
in concentration of about 0.1 to 10 percent w/v.
The invention has been described with reference to varius specific
and preferred embodiments and techniques. However, it should be
understood that many variations and modifications may be made while
remaining within the spirit and scope of the invention.
* * * * *